Method of diagnosing endometriosis in human subjects

ABSTRACT

A method for diagnosing endometriosis in a human subject comprising the steps of detecting a test amount of an antibody that specifically binds to ME-5 (SEQ ID NO:3) polypeptide or a peptide comprising an epitope of ME-5 in a sample from the subject; and comparing the test amount with a normal range of the antibody in a control sample from a subject who does not suffer from endometriosis, whereby a test amount above the normal range provides a positive indication in the diagnosis of endometriosis.

This is a divisional of pending application Ser. No. 11/593,874 filedNov. 6, 2006 which is a divisional of application Ser. No. 10/887,540filed Jul. 7, 2004, abandoned; the disclosures of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Endometriosis is a female reproductive disorder characterized by thepresence of endometrial tissue outside of the normal uterine location.Most frequently the endometriosis tissue is present in the peritonealcavity, attaching to various tissues and organs in this location.Endometriosis is a benign disease affecting approximately 5 millionwomen in the United States annually with a prevalence of 10 to 15percent in women of childbearing age. The incidence increases to 60 to80 percent of women who are infertile or present with pelvic pain (D.Gosselin et al. [1999] Curr. Opin. Onco. Endo. & Metabol. Invest. Drugs1:31). The conditions that predispose an individual to endometriosis arestill unknown. Several authoritative reports suggest that retrogrademenstruation may be a key-contributing factor, but this process isthought to be common in most women. This theory has also been questionedrecently (D. B. Redwine [2002] Fert. Steril. 78:686) due primarily tothe substantial differences that occur between normal or eutopicendometrium and the ectopic tissue found in diseased patients.Consequently, other genetic as well as immunological factors are thoughtto contribute key roles to the development of the disease in susceptiblewomen. For example, endometriosis is thought to be much more frequent infirst degree relatives of affected women when compared to the rest ofthe population (Coxhead and Thomas [1993] J. Obstet. Gynacol. 13:42). Inaddition to the frequency, the disease has also been reported to be moresevere in women with a first-degree relative with endometriosis (Thomasand Campbell [2000] Gynacol. Obstet. Invest. 50:2). The precise gene(s)involved in the disorder are unknown but the pattern is stronglysuspected to be maternal in nature.

Although not life threatening, endometriosis results in substantialabdominal discomfort, and may cause infertility. In fact, such symptomscan be indicative of other feminine health disorders and this makes thediagnosis of endometriosis clinically challenging. This was emphasizedin a recent study upon the effects of delayed diagnosis of endometriosis(G. K. Husby et al [2003] Acta. Obstet. Gynecol. Scand. 82:649). Theseinvestigators reported delays from 3 to 11 years between the onset ofpain and the final diagnosis of endometriosis. In this study womenreporting both infertility and pain did not have a significantly shorterdelay in diagnosis. Obviously such delays coupled with the symptomsreported lead to the expenditure of considerable economic andpsychological resources.

Currently surgical laparoscopy is considered to be the gold standard fordiagnosis of endometriosis. During laparoscopy the disease is visuallystaged using a point system from stage I (minimal disease, 1 to 5points) to IV (severe disease, >40 points). The points are assignedaccording to several parameters such as location, size, and depth(superficial versus deep) of the lesions (T. P. Canavan and L. Radosh[2000] Postgrad. Med. 107:213). Some opinions reveal potential hazardswith the procedure, and frequently laproscopy does not result in adefinitive diagnosis of the disease (S. Pillai et al. [1996] Am. J.Reprod. Immunol. 35:483). For example, while laparoscopy is not classedas major surgery it still has several features (invasive, expensive,requires anesthesia, and full operating facilities) which together makethe process an unfortunate choice for diagnosis at least. In fact, whileendometriosis is not a fatal disorder, laparoscopy itself can be lifethreatening. The trans-abdominal approach has been reported to beresponsible for 50% of the complications from this procedure, and injuryto major blood vessels can result in mortality of 15% (I. A. Brosens andJ. J. Brosens [2000] Eur. J. Obstet. Gynecol. Reprod. Biol. 88:117).Other complaints are that the visual staging of the disease does notcorrelate with the degree of infertility or the severity or number ofsymptoms (T. P. Canavan and L. Radosh [2000] Postgrad. Med. 107:213). Ithas been reported that the place of laparoscopy in the diagnosis ofendometriosis should be reassessed (I. A. Brosens and J. J. Brosens[2000] Eur. J. Obstet. Gynecol. Reprod. Biol. 88:117). Rational for thislies in part due to the suggestion by some (P. R. Koninckx [1994] Hum.Reprod. 9:2202) that mild endometriosis is not a disease at all and thatall women have endometriosis. Moreover as noted above there arefunctional aspects (e.g., infertility, abdominal pain, etc.) to stagesother than mild disease and these are more commonly being applied todiagnosis. Consequently it has been proposed that the traditional ‘goldstandard’ be replaced with a combination of transvaginalhydrolaparoscopy (THL, a somewhat milder procedure) and magneticresonance (MR) imaging until suitable biochemical markers have beenidentified (I. A. Brosens and J. J. Brosens [2000] Eur. J. Obstet.Gynecol. Reprod. Biol. 88:117).

The frequency of endometriosis and the difficulty of diagnosing thedisorder together represent ample rationale for experiments designed toidentify these serum based biochemical markers. Other discovery phaseprograms have implied that of potential markers of endometrial diseasemay exist. For example, levels of the epithelial ovarian-derived antigenCA-125 have been reported to be elevated in serum, peritoneal fluid, andmenstrual fluid of endometriosis patients (B. Mol et al. [1998] Fertil.Steril. 70:1101). The marker exhibited good specificity, but thesensitivity is poor with high levels present in patients afflicted withPID, ovarian cancer, or cervical carcinoma. Despite the limitations, themarker may be of use for patients who are likely to have the disease forfaster orientation toward laparoscopy, since CA-125 levels do correlatesomewhat with the degree of disease and response to treatment (T. P.Canavan and L. Radosh [2000] Postgrad. Med. 107:213).

Also, Sharpe-Timms et al. (Biol. Reprod. [1998] 58:988) have reportedthat endometriosis lesions secrete a haptoglobin-like protein in arodent model system. The haptoglobulin was specifically synthesized byendometriosis tissue and was not found in uterine tissue using asensitive reverse transcriptase PCR technique. This antigen is alsointeresting in that it has been reported to modulate immune cellfunctions and could contribute to the pathophysiology of endometriosis

Along slightly different lines, D. Gosselin et al. (Curr. Opin. Oncol.Endo. Metabol. Inv. Drugs [1999] 1:31) reported a diagnostic algorithmemploying several different combinations of leukocyte markers presentupon subsets of T and B cells, macrophages, and NK cells in peripheralblood and endometrium of patients with endometriosis. This formed thefoundation for the development of a diagnostic test (Metrio Test) byPROCREA BioSciences, Inc. which is approved by Health Canada. The MetrioTest is based on the assessment of eight proprietary leukocyte subsetsby flow cytometry analysis combined with a blood biochemical markerevaluated by ELISA (J. Brosens et al. Obstet. Gynecol. Clin. North Am.[2003] 30:95-114). This test reportedly has a specificity rate of 95%and a sensitivity rate of 61%.

P. Vigano et al. (Obstet. Gynecol. [2000] 95:115-118) report that thesoluble form of intercellular adhesion molecule 1 is released by uterineendometrium and such release correlates with the extent (number ofimplants) of endometriosis in patients. The authors suggest that solubleintercellular adhesion molecule 1 might be of value in evaluating thespread potential of refluxed endometrium. However, soluble intercellularadhesion molecule 1 is also known to be released in other disease statesso the potential value of this protein as a marker may be diminishedsomewhat.

J. Mahnke et al. (Fertil. Sterl. [2000] 73:166-170) evaluated VEGF andIL-6 levels in peritoneal fluid of women with endometriosis and foundthem to be elevated in patients with advanced disease. The levels ofVEGF and IL-6 were lower in normal women and patients with milderdisease. Nevertheless, the diagnostic value of these markers is suspectsince at least VEGF is known to be a potent angiogenesis factor that isregulated by hypoxia in normal endometrium (A. M. Sharkey et al. J.Clin. Endocrinol. Metab. [2000] 85:402-409).

Matalliotakis and coworkers (Obstet. Gynecol. [2000] 95:810-813) foundelevated levels of soluble CD23 in serum of women with endometriosiswhen compared to a control population. The CD23 levels decreasedsignificantly during treatment with either danazol or leuprolideacetate. There seemed to be no correlation between soluble CD23 levelsand the severity of endometriosis in the patients. As noted above forsome of the other putative markers, CD23 has been associated withconditions linked to autoantibody production and levels of this proteinare elevated in patients with autoimmune diseases.

Overall despite the substantial effort extended by numerous researchers,and also as reported in the publications reviewed above, no trulyacceptable marker for endometriosis has been discovered. Yet, thephysical and economic impact of the disease, and the difficulty indiagnosing the disorder dictate that the search for suitable markers becontinued. Consequently, the activities disclosed in this invention wereundertaken to identify markers of endometriosis that can aid physiciansin monitoring patients with this illness. Other groups have performedsuch projects and these discoveries are the subject of numerous patentdocuments, which differ substantially from the discovery of the ME-5,ME-2 and EPP2 markers described in this invention. In U.S. Patentapplication 2003/0032044 there is a description of methods for generallydetecting reproductive tract disorders by measuring the levels ofinterleukins IL-13 and IL-15 in specimens. Another U.S. Patentapplication 2002/0192647 proposes a process for diagnosing angiogenicdiseases by measuring a single nucleotide polymorphism in the VEGFR-1gene. Endometriosis is categorized as one of this group of angiogenicdiseases, but it was not the subject of any of the claims. Patentapplications 2001/046713 and 2001/044158 describe a method for diagnosisof endometriosis by detecting anti-Tomsen-Frienenreich antibodies inspecimens. An issued U.S. Pat. No. 6,376,201 illustrates the use ofmajor histocompatibility complex-class I antigens in diagnosingendometriosis and forming the basis of the Metrio Test as describedabove. In this patent the MHC-class I antigens are detected in specimenswith specific monoclonal antibodies and similar disclosures weredescribed in U.S. Pat. No. 5,618,680 and W.O. 0043789. A method fordiagnosing endometriosis is described in U.S. Pat. No. 6,540,980 thatinvolves measurement of eosinophil peroxidase levels. In U.S. Pat. No.6,525,187 is described an apparently novel, marker of endometriosiswhich is the target of autoantibodies present in patient serum. Anothermethod for diagnosis of endometriosis is disclosed in U.S. Pat. No.6,387,629 and this is based upon the measurement of the proteasecathepsin S in a clinical sample. A gene encoding an endometrialbleeding associated factor (ebaf) is described in U.S. Pat. No.6,294,662 and this gene could be useful for diagnosis of endometriosis.However the ebaf gene seems to have better utility in the earlydiagnosis of selected carcinomas (colon, ovaries, or testis) in a human.In U.S. Pat. No. 5,877,284 another potential marker of endometriosis isdescribed. This marker is a small soluble protein isolated by affinitychromatography from the peritoneal fluid of women with endometrosis, andthe protein has chemotactic activity to neutrophils and macrophages. Aprocess for monitoring human endometrial functions is described in U.S.Pat. No. 4,489,166 and it involves the quantitative measurement ofprogestagen-associated endometrial protein (PEP) in a clinical sample.European Patent No. 1191107 describes a method for diagnosis ofendometriosis by measuring a reduction in the levels of one of a groupof 15 different human genes. An immunoassay process is described inEuropean Patent No. 0387027 which establishes endometriosis in a patientby evaluating a specimen with an anti-endometriosis monoclonal antibody.A method is described in W.O. 0063675 for diagnosis of endometriosis bymeasuring increased levels of endometriosis factor in biological fluidsof a patient. W.O. 9963116 provides for a method of diagnosingendometriosis by measuring increases in the amount of prothymosin inendometriotic tissue.

U.S. Pat. No. 6,531,277 discloses an endometriosis-specific secretoryprotein. The document characterized and disclosed human ENDO-1 that isproduced by stromal cells of endometriotic tissue. The ENDO-1 protein is40 to 50 kilodaltons in molecular weight and has an isoelectric point of4.0 to 5.5. The claims of the document are concerned primarily with amolecular diagnostic assay measuring differences in expression of ENDO-1mRNA in endometrosis tissue samples. In a related application U.S.2002/0009718 the invention is extended for measurement of the ENDO-1glycoprotein in patient samples using immunoassay to establish thepresence of endometriosis. Nevertheless, the characteristics of ENDO-1presented in these documents suggest that it is considerably differentfrom the markers described in the present invention. For example whenmeasured by SDS PAGE and Western blotting the ME-5, ME-2, and EPP2proteins are about 38, 49, and 9 kilodaltons in size, respectively. Onlythe ME-2 marker is within the range specified for ENDO-1, but ME-2 hasan isoelectric point of 8.8 so it is not a related protein. Also, theisoelectric points of the ME-5 and EPP2 antigens are calculated at 5.7and 12.5, respectively, which are also well above the range of valuesspecified for the ENDO-1 protein. Moreover the ENDO-1 marker is a memberof the haptogloblin family of proteins, but nucleic acid and amino acidsequence comparisons show that the ME-5, ME-2, and EPP2 markers are notrelated to this family of proteins.

In yet another separate disclosure, U.S. Pat. No. 5,843,673 specifies amethod of screening for endometriosis in women by measuring a reductionin the amounts of a 28 to 32 kilodalton molecular weight glycoprotein inperitoneal fluid or serum samples. The protein possesses an isoelectricpoint of 7.0 to 9.0 and is secreted specifically by stromal cells ofendometriotic origin. The glycoprotein disclosed in the document isrelated to tissue inhibitor of metaloproteinases-1 (TIMP-1) by virtue ofamino acid sequence identity measured in the amino terminal region ofprotein. In the patent it is shown that endometriosis is indicated in apatient who has reduced levels of TIMP-1 present in serum or peritonealfluid. The ME-5, ME-2, and EPP2 proteins of this invention are notrelated to TIMP-1 and they have no measurable protein or nucleic acidhomology to this family of proteins. In addition, and as noted above,the biochemical properties of the ME-5, ME-2, and EPP2 proteins differfrom those of TIMP-1 and each is considerably larger or smaller (at 38,49, or 9 kilodaltons, respectively) than the range given for TIMP-1.While the isoelectric point of ME-2 is at the upper range of that ofTIMP-1, the isoelectric point of ME-5 is 5.7 and EPP2 is 12.5 which aremuch different.

Another disclosure of protein agents implicated in endometriosis iscontained in the document WO 01/32920 in which it is assumed that atotal of 33 genes and their protein products are associated with thedisease. These putative endometriosis markers were identified bycomparing the pattern of gene expression in diseased endometriumrelative to that of normal tissue. This differential display reversetranscriptase polymerase chain reaction employed in the document is apurely genetic screening approach designed to identifydisease-associated genes based upon differences in the expression levelsof mRNAs. The mRNA populations compared are usually normal healthyendometrium and the diseased counterpart, ideally both isolated from asingle patient suffering from the illness. This technology ignores thefunctional activity of the proteins encoded by the mRNAs, and does notinterrogate specimens based on disease hallmarks, symptoms, or thebody's response to the illness. The latter strategies are arguablybetter approaches for marker discovery as discussed below. Theindividual nucleic acid sequences identified in the document fall intothe general groups of; protease or protease inhibitor, tumor suppressorprotein, immune system proteins, inflammatory response proteins,enzymes, lipid binding proteins, transcription factors, and matrix orcell adhesion molecules. All of the genes in WO 01/32920 are known andthe nucleic acid sequences appear in the public databases allowing themto be identified. The individual nucleic acid sequences identified andimplicated as somehow being involved in endometriosis are: cathepsin D,AEBP-1, stromelysin-3, cystatin B, protease inhibitor 1, sFRP4,gelsolin, IGFBP-3, dual specificity phosphatase 1, PAEP, immunoglobulinA chain, ferritin, complement component 3, pro-alpha-1 type IIIcollagen, proline 4-hydroxylase, alpha-2 type I collagen, claudin-4,melanoma adhesion protein, procollagen C-endopeptidase enhancer,nascent-polypeptide-associated complex alpha polypeptide, elongationfactor 1 alpha (EF-1a), vitamin D3 25 hydroxylase, CSRP-1, steroidogenicacute regulatory protein, apolipoprotein E, transcobalamin II,prosaposin, early growth response 1 (EGR1), ribosomal protein S6,adenosine deaminase RNA-specific protein, RAD21, guanine nucleotidebinding protein beta polypeptide 2-like 1 (RACK1), and podocalyxin (andsee references within WO 01/32920). Overall the diagnosis ofendometrosis with the above agents would involve assessing the level ofexpression of the gene. The ME-5, ME-2, and EPP2 proteins and thenucleic acids described in this invention are also known and appear inthe databases (see Example 1, below). However, none of the ME-5, ME-2,or EPP2 sequences fall into any of the groups listed above nor do theycorrespond to any of the designated agents either by computer-assistedhomology comparison or predicted function based upon the presence ofrecognizable motifs present in the protein sequence. A similar geneexpression-based strategy was employed in the discoveries documented byS. Baban et al. in US Patent Application 2002/0127555 in which 14 geneswere found to be overexpressed in endometriosis patients relative todisease-free females. The overexpressed genes were NADH dehydrogenase,hUCCi, Paralemmin, citrate transport protein. HIF1-alpha, ARNT, Glut-1,MnSOD, GPx, ATP synthase, c-jun, Cx43, HSP 70, and cox2. In addition, 19genes were reported in this document to be underexpressed inendometriosis patients relative to disease-free females. The genesunderexpressed in diseased endometrial tissues were Cap43, RNA helicase,CO3, FKHR, AK3, catalase, GST, eNOS, 12S rRNA, T1227H, CO2, aconitase,ANT-1, Bcl-2, COUP-TF, IL-1 beta, HSP 90, GPx4, and GRP78. Yet anothergene expression strategy was described by H. Hess-Stumpp et al. In USPatent Application 2003/0077589 resulting in the discovery of 15 genesthat are overexpressed in endometriosis. The overexpressed genes werefibronectin, IGFBP-2, transmembrane receptor PTK7, platelet-derivedgrowth factor alpha, collagen type XVIII alpha 1, subtilisin-likeprotein (PACE4), laminin M chain (merosin), elastin, collagen type IValpha 2, p27interferon alpha-inducible gene, reticulocalbin, aldehydedehydrogenase 6, gravin, nidogen, and phospholipase C epsilon. Again, asstated above, the ME-5, ME-2, and EPP2 protein and nucleic acidsequences are not related to any of the genes described in the lattertwo patents.

Taken together and comparing the results of these three documents, it isinteresting that all of them used similar but not identical geneexpression strategies to identify a total of 62 genes which areoverexpressed in endometriosis and 19 genes that are underexpressed. Theimplication is therefore that the 81 described genes are related to orinvolved in endometrial disease. Surprisingly, among these threeindependent studies, no single human gene or class of genes wasconsistently found to be associated with endometriosis. Ostensibly if agene were overexpressed because of changes occurring in endometriosistissue relative to the normal counterpart, then it would be expected toreproducibly be identified in all studies that assess the geneexpression profile of diseased tissue. This does not seem to occur inthe otherwise well-designed projects, and brings into questionstrategies for marker discovery based only on gene expression profilingtechnologies.

The document WO 94/28021 describes endometrial proteins, antigeniccompounds, and methods of detecting endometriosis. The disclosureencompasses endometriosis-specific proteins defined by molecular weightand isoelectric point. Many of the claims presented are based only onsize, but others specify a molecular weight and isoelectric point. Theprincipal endometriosis antigen of the document and which is describedin the initial claim has a molecular weight of 64 kilodaltons and anisoelectric point of 3.5. The antigen is used to measure antibodies inspecimens obtained from endometriosis patients and also can itself bemeasured directly for its presence in patient samples. In addition, alarger molecular weight endometriosis protein of 94 kilodaltons with anisoelectric point of 3.5 is also described presumably to be used in thesame formats as the smaller antigen. The document also claims nucleicacids for these. proteins, however these sequences do not appear inenough detail to allow for comparison to the ME-5, ME-2, and EPP2protein and nucleic acids of this invention. A small amount of aminoacid sequence is presented in WO 94/28021, but there are only 17residues shown in the document and of these over half are ambiguous.Although similar applications are envisioned for the ME-5, ME-2, andEPP2 protein described in this invention, the antigens described abovedo not compare in any reported properties to those of the threeendometrosis antigens presented here. Initially, none of the unambiguousresidues of amino terminal protein sequence are present in thecorresponding regions of ME-5, ME-2, and EPP2. In addition, the ME-5,ME-2, and EPP2 proteins are 38, 49, and 9 kilodaltons in size, which areconsiderably smaller than the antigens described in the documentoutlined above. Moreover the isoelectric points of ME-5, ME-2, and EPP2are 5.7, 8.8, and 12.5 which are considerably greater than described forthe other proteins. It must be concluded that the endometrial ME-5,ME-2, and EPP2 antigens of this invention have little in common with theproteins described in WO 94/28021.

Methods and reagents for diagnosis of endometriosis are described in NZ232801 (also application EP-A-0 387 027) essentially by measuring anendometriosis antigen in a patient specimen using an anti-endometriosisantibody. Various antigens are described in the document ranging inmolecular weight from 50 to 173 kilodaltons but no additionalcharacterization of the proteins was performed. These proteins wereisolated as a mixture from the culture medium and cytoplasm of 2774ovarian carcinoma cells, and can be obtained from other cultured celllines as well. Also described in the disclosure is an anti-endometrialantibody, which is a human IgM monoclonal originally isolated because itreacted with ovarian cancer-associated antigens. Isolation of theantibody was apparently through a set of activities that were unrelatedto endometriosis and the ovarian cancer antigen targets apparently werenot well characterized. The antibody was made by fusion of patientlymphocytes with a heteromyeloma, and apparently the reactivity of themonoclonal with endometrial antigens was discovered subsequently.Regardless, based on the criteria presented it is unlikely that any ofthe proteins of NZ 232801 are the same as the smaller ME-5, ME-2, andEPP2 proteins of this invention.

Another series of endometrial antigens reactive with anti-endometrialantibodies is described in WO 92/18535 and these are also characterizedby molecular weight on SDS PAGE analysis. The described protein antigenfragments were isolated from the cytoplasm of epithelial adenocarcinomacells and are described as useful for detection of endometrialantibodies which are indicative of endometriosis. The antigens arecytoplasmic proteins with sizes of 63 to 67, 33 to 37, 40 to 44, 31 to35, and 57 to 64 kilodaltons. The designations likely refer to a singleprotein species, but the size ranges were presented in the document toreflect the inherent inaccuracy (±10%) for the SDS PAGE assay methodused. Apparently the preferred proteins for use are the 33 to 37, 40 to44, and the 57 to 59 kilodalton proteins. The 33 to 37 and 40 to 44proteins seemed to be present in most of the cell lines that werestudied in the document for use as sources of antigen, while the 57 to59 protein fragments originates from the T47D breast carcinoma cellline. The document describes the use of these proteins individually (ormixed) immobilized on solid support to measure endometrial antibodies.Of course similar applications are envisioned for the ME-5, ME-2, andEPP2 antigens, however with the exception of possibly the 33 to 37kilodalton fragments there is little else presented in this documentthat compares to disclosures in WO 92/18535.

SUMMARY OF THE INVENTION

A recombinant polynucleotide comprising an isolated nucleotide sequencefrom SEQ ID NO:2 encoding a polypeptide epitope of at least 5 aminoacids of ME-5 (SEQ ID NO:3), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A purified, recombinant ME-5 polypeptide whose amino acid sequence issubstantially identical to that of SEQ ID NO:3 or an allelic variant ofSEQ ID NO:3.

A purified polypeptide comprising an epitope of at least 5 amino acidsof ME-5 (SEQ ID NO:3), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A composition consisting essentially of an antibody that specificallybinds to an epitope of ME-5 polypeptide (SEQ ID NO:3).

A method for detecting a ME-5 polypeptide (SEQ ID NO:3) in a sample,comprising the steps of:

-   -   (a) contacting the sample with an antibody that specifically        binds to an epitope of the ME-5 polypeptide and    -   (b) detecting specific binding between the antibody and ME-5        polypeptide;        whereby specific binding provides a detection of ME-5        polypeptide in the sample.

A method for diagnosing endometriosis in a human subject comprising thesteps of:

-   -   (a) detecting a test amount of an antibody that specifically        binds to an epitope of ME-5 polypeptide (SEQ ID NO:3) in a        sample from the subject; and    -   (b) comparing the test amount with a normal range of the        antibody in a control sample from a subject who does not suffer        from endometriosis,        whereby a test amount above the normal range provides a positive        indication in the diagnosis of endometriosis.

A recombinant polynucleotide comprising an isolated nucleotide sequencefrom SEQ ID NO:5 encoding a polypeptide epitope of at least 5 aminoacids of ME-2 (SEQ ID NO:6), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A purified, recombinant ME-2 polypeptide whose amino acid sequence isidentical to that of SEQ ID NO:6 or an allelic variant of SEQ ID NO:6.

A purified polypeptide comprising an epitope of at least 5 amino acidsof ME-2 (SEQ ID NO:6), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A composition consisting essentially of an antibody that specificallybinds to an epitope of ME-2 polypeptide (SEQ ID NO:6).

A method for detecting a ME-2 polypeptide (SEQ ID NO:6) in a sample,comprising the steps of:

-   -   (a) contacting the sample with an antibody that specifically        binds to an epitope of the ME-2 polypeptide and    -   (b) detecting specific binding between the antibody and ME-2        polypeptide;        whereby specific binding provides a detection of ME-2        polypeptide in the sample.

A method for diagnosing endometriosis in a human subject comprising thesteps of:

-   -   (a) detecting a test amount of an antibody that specifically        binds to an epitope of ME-2 polypeptide (SEQ ID NO:6) in a        sample from the subject; and    -   (b) comparing the test amount with a normal range of the        antibody in a control sample from a subject who does not suffer        from endometriosis,        whereby a test amount above the normal range provides a positive        indication in the diagnosis of endometriosis.

A recombinant polynucleotide comprising an isolated nucleotide sequencefrom SEQ ID NO:8 encoding a polypeptide epitope of at least 5 aminoacids of EPP2 (SEQ ID NO:9), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A purified, recombinant EPP2 polypeptide whose amino acid sequence isidentical to that of SEQ ID NO:9 or an allelic variant of SEQ ID NO:9.

A purified polypeptide comprising an epitope of at least 5 amino acidsof EPP2 (SEQ ID NO:9), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

A composition consisting essentially of an antibody that specificallybinds to an epitope of EPP2 polypeptide (SEQ ID NO:9).

A method for detecting a EPP2 polypeptide (SEQ ID NO:9) in a sample,comprising the steps of:

-   -   (a) contacting the sample with an antibody that specifically        binds to an epitope of the EPP2 polypeptide and    -   (b) detecting specific binding between the antibody and EPP2        polypeptide;        whereby specific binding provides a detection of EPP2        polypeptide in the sample.

A method for diagnosing endometriosis in a human subject comprising thesteps of:

-   -   (a) detecting a test amount of an antibody that specifically        binds to an epitope of EPP2 polypeptide (SEQ ID NO:9) in a        sample from the subject; and    -   (b) comparing the test amount with a normal range of the        antibody in a control sample from a subject who does not suffer        from endometriosis,        whereby a test amount above the normal range provides a positive        indication in the diagnosis of endometriosis.

A composition containing at least one of

a purified, recombinant ME-5 polypeptide whose amino acid sequence issubstantially identical to that of SEQ ID NO:3 or an allelic variant ofSEQ ID NO:3;

a purified, recombinant ME-2 polypeptide whose amino acid sequence issubstantially identical to that of SEQ ID NO:6 or an allelic variant ofSEQ ID NO:6; and

a purified, recombinant EPP2 polypeptide whose amino acid sequence issubstantially identical to that of SEQ ID NO:9 or an allelic variant ofSEQ ID NO:9.

A composition containing at least one of a purified polypeptidecomprising an epitope of at least 5 amino acids of ME-5 (SEQ ID NO:3);

a purified polypeptide comprising an epitope of at least 5 amino acidsof ME-2 (SEQ ID NO:6), and

a purified polypeptide comprising an epitope of at least 5 amino acidsof EPP2 (SEQ ID NO:9),

wherein said epitopes specifically bind to antibodies from subjectsdiagnosed with endometriosis.

A method for diagnosing endometriosis in a human subject comprising thesteps of:

-   -   (a) detecting a test amount of an antibody that specifically        binds to at least one of ME-5 (SEQ ID NO:3) polypeptide, ME-2        (SEQ ID NO:6) polypeptide; and EPP2 (SEQ ID NO:9) polypeptide in        a sample from the subject; and    -   (b) comparing the test amount with a normal range of the        antibody in a control sample from a subject who does not suffer        from endometriosis,        whereby a test amount above the normal range provides a positive        indication in the diagnosis of endometriosis.

As shown in the documents cited above, a number of discoveries have beendocumented for candidate markers of endometriosis. None of thosecorrespond to the ME-5, ME-2, or EPP2 proteins and nucleic acidsequences of the present invention. Consequently the ME-5, ME-2, andEPP2 proteins of this invention represent novel new markers forendometriosis and the targets of anti-endometrial antibodies produced bywomen suffering from the disorder. The discovery of the ME-5, ME-2, andEPP2 markers of this invention was predicated upon the knowledge thatwomen suffering from endometriosis have defects in their immune systems.It is assumed that some immune system problems may be manifest in thepresence of autoantibodies directed towards endometrial antigens. Others(S. Pillai et al. [1998] Am. J. Reprod. Immunol. 39:235; Van Voorhis andStovall [1997] J. Reprod. Immunol. 33:239) have discussed such asituation. Clearly, this represents an attractive means of identifyingcandidate markers of the disease and as useful tools for monitoringpatients with endometriosis. Recently, a summary of the accuracy ofserum markers for the diagnosis of endometriosis showed endometrialantibodies to be among the best markers with sensitivity of 74% to 83%and specificity of 79% to 100% (J. Brosens et al. 2003] Obstet. Gynecol.Clin. North Am. 30:95). However the antibodies were not measured againstdiscrete isolated antigens such as ME-5, ME-2, and EPP2 for example.

In initiating a program to identify antigens that may be useful markersof endometriosis (and thus helpful in monitoring women that suffer thedisorder) some assumptions were made regarding this disease. First, asnoted above, it was assumed that immune system defects occur in thesewomen which enable them to make antibodies directed towards specificendometrial antigens. Second these serum antibodies could be used astools to identify the antigens, and these proteins in part would formthe foundation of immunodiagnostic test systems for monitoring patientswith the disorder. The strategy for identification of endometriosismarkers was to use patient serum to immunoscreen an endometrial tissuecDNA expression library. Candidate clones would be completelycharacterized for development of an immunoassay suitable for monitoringpatients in a clinical environment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A total of three endometrial proteins are described that react withantibodies present in the serum of endometriosis patients. The ME-5endometriosis marker is specified by a mRNA of about 1.4 kb, of which1,302 nucleotides is disclosed in this invention. The protein predictedfrom this sequence is 303 amino acids in size and has a calculatedmolecular weight of about 35,000 daltons. The natural protein producthas a molecular weight of about 38 kD as measured by Western blot with aspecific monoclonal antibody. The protein was particularly abundant inovary tissue which, taken with the isolation from endometrial tissue isstrongly supportive of its presence in reproductive tissues and as amarker of reproductive disease. In immunoblotting experiments withimmobilized recombinant ME-5 antigen, a number of endometriosis patientswere evaluated and the signals generated were considerably stronger thanthat obtained with a number of control patients.

The ME-2 endometriosis marker is specified by a mRNA of about 2.0 kb ofwhich 1,353 nucleotides is disclosed in this invention. The proteinpredicted from this sequence is 393 amino acids in size and has acalculated molecular weight of about 45,000 Daltons. In immunoblottingexperiments with immobilized recombinant ME-2 antigen evaluated with anumber of endometriosis patients the signal generated was considerablystronger than that obtained with a number of control patients.

The EPP2 endometriosis marker is specified by a mRNA of about 1.0 kb ofwhich 891 nucleotides is disclosed in this invention. The proteinpredicted from this sequence is 99 amino acids in size and has acalculated molecular weight of about 9,300 Daltons. In immunoblottingexperiments with immobilized recombinant EPP2 antigen evaluated with anumber of endometriosis patients the signal generated was considerablystronger than that obtained with a number of control patients.

Details of these and other issues related to the ME-5, ME-2, and EPP2endometriosis markers and their nucleic acids are contained in theexamples below.

Clearly as cited by the documents presented above, a number ofdiscoveries have been documented for candidate markers of endometriosis.None of those correspond to the ME-5, ME-2, or EPP2 proteins and nucleicacid sequences disclosed herein. Consequently the ME-5, ME-2, and EPP2proteins of this invention represent novel new markers for endometriosisand the targets of anti-endometrial antibodies produced by womensuffering from the disorder. The discovery of the ME-5, ME-2, and EPP2markers in this invention was predicated upon the knowledge that womensuffering from endometriosis have defects in their immune systems. It isassumed that some immune system problems may be manifest in the presenceof autoantibodies directed towards endometrial antigens. Others (S.Pillai et al. [1998] Am. J. Reprod. Immunol. 39:235; Van Voorhis andStovall [1997] J. Reprod. Immunol. 33:239) have discussed such asituation. Clearly, this represents an attractive means of identifyingcandidate markers of the disease and as useful tools for monitoringpatients with endometriosis. Recently, a summary of the accuracy ofserum markers for the diagnosis of endometriosis showed endometrialantibodies to be among the best markers with sensitivity of 74% to 83%and specificity of 79% to 100% (J. Brosens et al. 2003] Obstet. Gynecol.Clin. North Am. 30:95). However the antibodies were not measured againstdiscrete isolated antigens such as ME-5, ME-2, and EPP2 for example.

In initiating a program to identify antigens that may be useful markersof endometriosis (and thus helpful in monitoring women that suffer thedisorder) some assumptions were made regarding this disease. First, asnoted above, it was assumed that immune system defects occur in thesewomen which enable them to make antibodies directed towards specificendometrial antigens. Second these serum antibodies could be used astools to identify the antigens, and these proteins in part would formthe foundation of immunodiagnostic test systems for monitoring patientswith the disorder. The strategy for identification of endometriosismarkers was to use patient serum to immunoscreen an endometrial tissuecDNA expression library. Candidate clones would be completelycharacterized for development of an immunoassay suitable for monitoringpatients in a clinical environment.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1) for theisolated ME-5 cDNA, the nucleotide sequence of the coding region (SEQ IDNO:2) of this ME-5cDNA, and the deduced amino acid sequence (SEQ IDNO:3) of the protein encoded by the nucleotide sequence of the ME-5cDNA. In FIG. 1A there is a 112 base pair 5′ untranslated sequenceupstream of the predicted ATG start codon. Also in FIG. 1A is a 254 basepair 3′ untranslated region downstream of the TGA stop codon. The 3′untranslated region terminates at a stretch of dT corresponding to thepoly A tail of the mRNA. The start Codon (ATG) and the translation stopcodon (TGA) are presented in bold type in the cDNA sequence of FIGS. 1Aand B.

FIGS. 2A, 2B, and 2C show the nucleotide sequence (SEQ ID NO:4) for theisolated ME-2 cDNA, the nucleotide sequence of the coding region (SEQ IDNO:5) of this ME-2 cDNA, and the deduced amino acid sequence (SEQ IDNO:6) of the protein encoded by the nucleotide sequence of the ME-2cDNA. In FIG. 2A there is a 54 base pair 5′ untranslated sequenceupstream of the predicted ATG start codon. Also in FIG. 2A is a 95 basepair 3′ untranslated region downstream of the TAG stop codon. The 3′untranslated region terminates at a stretch of dT corresponding to thepoly A tail of the mRNA. The start codon (ATG) and the translation stopcodon (TAG) are presented in bold type in the cDNA sequence of FIGS. 2Aand B.

FIGS. 3A, 3B, and 3C show the nucleotide sequence (SEQ ID NO:7) for theisolated EPP2 cDNA, the nucleotide sequence of the coding region (SEQ IDNO:8) of this EPP2 cDNA, and the deduced amino acid sequence (SEQ IDNO:9) of the protein encoded by the nucleotide sequence of the EPP2cDNA. In FIG. 3A there is a 45 base pair 5′ untranslated sequenceupstream of the predicted ATG start codon. Also in FIG. 3A is a 522 basepair 3′ untranslated region downstream of the TAA stop codon. The 3′untranslated region terminates at a stretch of dT corresponding to thepoly A tail of the mRNA. The start codon (ATG) and the translation stopcodon (TAA) are presented in bold type in the cDNA sequence of FIGS. 3Aand B.

FIG. 4 demonstrates the pattern of ME-5 mRNA expression in various humantissues. A commercial Northern blot (BD Biosciences; San Diego, Calif.)was hybridized with the complete ³²P-labeled ME-5 coding sequence ofFIG. 1B. Conditions of hybridization and washing were as described bythe manufacturer. Hybridizing bands were observed corresponding to amRNA of about 1,400 nucleotides (migrates just slower than the 1,350nucleotide marker) as well as another larger but perhaps less abundantmessage of 1,800 to 2,000 nucleotides (migrating just ahead of the 2,400nucleotide marker). The ME-5 sequence seems to be expressed mostabundantly in prostate, testis and uterus tissues, but lower amountswere detected in the other tissues evaluated (spleen, thymus, smallintestine, colon and peripheral blood leukocyte).

FIG. 5 demonstrates the pattern of ME-2 mRNA expression in various humantissues. A commercial Northern blot (BD Biosciences; San Diego, Calif.)was hybridized with the complete ³²P-labeled ME-2 coding sequence ofFIG. 2B. Conditions of hybridization and washing were as described bythe manufacturer. Hybridizing bands were observed corresponding to amRNA of about 2,000 nucleotides (migrates about mid way between the2,400 nucleotide and the 1,350 nucleotide markers). No other stronglyhybridizing bands were detected upon the blot. The ME-2 sequence seemsto be expressed most abundantly in prostate and testis tissues. Moderatelevels are detectable in spleen, uterus, small intestine, colon, andperipheral blood leukocyte tissues. In this experiment lower amounts ofhybridization were observed in thymus tissue.

FIG. 6 demonstrates the pattern of EPP2 mRNA expression in various humantissues. A commercial Northern blot (BD Biosciences; San Diego, Calif.)was hybridized with the complete ³²P-labeled EPP2 coding sequence ofFIG. 3B. Conditions of hybridization and washing were as described bythe manufacturer. Hybridizing bands were observed corresponding to amRNA of about 1,000 nucleotides (migrates just faster than the 1,350nucleotide marker). The EPP2 sequence seems to be expressed mostabundantly in prostate, testis, colon and peripheral blood leukocyte.Lesser amounts of signal were visualized in spleen, thymus, and smallintestine tissues, but little or no signal was detected in uterustissue.

FIG. 7 shows the pattern of expression of recombinant ME-5 in an insectcell host. The ME-5 cDNA was cloned for expression as a 6×histidine-tagged recombinant protein in insect cells. A culture of Sf9insect cells expressing recombinant ME-5 was prepared and lysed. Theculture medium, PBS wash, and the soluble and insoluble fractions of thecell lysate were analyzed by SDS PAGE and staining (left panel) of thegel with GelCode blue (Pierce Chemicals; Rockford, Ill.). The expressionsamples were also evaluated by Western blotting (right panel) with ananti-HisG mouse monoclonal antibody (Invitrogen; Carlsbad, Calif.)followed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody.The recombinant protein was obscured by the multiplicity of proteinbands in the stained gel at left, but a band of about 38 kD was clearlydetected by the Western blot. This confirmed the presence of a6×His-tagged protein with the approximate molecular weight expected forthe recombinant ME-5 antigen. No recombinant ME-5 protein was detectablein the cell culture medium, but some was present in the PBS used to washthe insect cells prior to lysis. Most of the recombinant ME-5 proteinseemed to be present in the soluble fraction of the insect cell lysate,but some was associated with the insoluble material.

FIG. 8 shows the pattern of expression of recombinant ME-2 in an insectcell host. The ME-2 cDNA was cloned for expression as a 6×histidine-tagged recombinant protein in insect cells. A culture of Sf9insect cells expressing recombinant ME-2 was prepared and lysed. Theculture medium, PBS wash, and the soluble and insoluble fractions of thecell lysate were analyzed by SDS PAGE and staining (left panel) of thegel with GelCode blue (Pierce Chemicals; Rockford, Ill.). The expressionsamples were also evaluated by Western blotting (right panel) with ananti-HisG mouse monoclonal antibody (Invitrogen; Carlsbad, Calif.)followed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody.The recombinant protein was obscured by the multiplicity of proteinbands in the stained gel at left, but a band of about 49 kD was clearlydetected by the Western blot. This confirmed the presence of a6×His-tagged protein with the approximate molecular weight expected forthe recombinant ME-2 protein. No recombinant ME-2 protein was detectablein the cell culture medium, but some was present in the PBS used to washthe insect cells prior to lysis. Approximately equal amounts of therecombinant ME-2 protein seemed to be distributed between the solubleand the insoluble fractions of the insect cell lysate.

FIG. 9 shows the pattern of expression of recombinant EPP2 in an insectcell host. The EPP2 cDNA was cloned for expression as a 6×histidine-tagged recombinant protein in insect cells. A culture of Sf9insect cells expressing recombinant EPP2 was prepared and lysed. Theculture medium, PBS wash, and the soluble and insoluble fractions of thecell lysate were analyzed by SDS PAGE and staining (left panel) of thegel with GelCode blue (Pierce Chemicals; Rockford, Ill.). The expressionsamples were also evaluated by Western blotting (right panel) with ananti-HisG mouse monoclonal antibody (Invitrogen; Carlsbad, Calif.)followed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody.The recombinant protein was obscured by the multiplicity of proteinbands in the stained gel at left, but a band of about 9 kD was clearlydetected by the Western blot. This confirmed the presence of a6×His-tagged protein with the approximate molecular weight expected forthe recombinant EPP2 protein. No recombinant EPP2 protein was detectablein the cell culture medium, nor was any measurable amount present in thePBS used to wash the insect cells prior to lysis. Approximately equalamounts of the recombinant EPP2 protein seemed to be distributed betweenthe soluble and the insoluble fractions of the insect cell lysate.

FIG. 10 shows the isolation of the recombinant 6×-tagged ME-5 proteinusing immobilized metal affinity chromatography (IMAC). Recombinant ME-5protein was expressed in Sf9 insect cells and the cells were lysed inIMAC column binding buffer. The soluble fraction of the insect cells(Lysate) was loaded onto a column of Chelating Sepharose Fast Flow(Amersham Biosciences; Piscataway, N.J.) that had been charged withnickel ions. The lysate was captured after passing through the columnresin (breakthrough) and the column was washed extensively with IMACwash buffer. The recombinant ME-5 bound to the resin was eluted from thecolumn with buffer containing imidazole. Samples of the lysate,breakthrough, wash, and elution were analyzed by SDS PAGE and Westernblot as described above. The stained gel showed the complexity of theinsect cell lysate, which resulted in a smear of protein for this andthe breakthrough samples. A reasonable amount of non-binding proteincontaminants were washed away with the A20 Column buffer, and a niceband corresponding to a 38 kD protein was present among the materialeluted from the column with imidazol. Western blotting of these samplesshowed good levels of the recombinant ME-5 protein in the lysate, and inthe breakthrough showing that in this particular experiment the amountof ME-5 exceeded the binding capacity for the column. Perhaps a trace ofME-5 was in the A20 Column buffer wash used to remove bound impuritiesfrom the Sepharose. The Western showed intense anti-HisG antibodyreactivity with the eluted and partially purified 38 kD ME-5 antigen.

FIG. 11 shows the isolation of the recombinant 6×-tagged ME-2 proteinusing immobilized metal affinity chromatography (IMAC). Recombinant ME-2protein was expressed in Sf9 insect cells and the cells were lysed inIMAC column binding buffer. The soluble fraction of the insect cells(Lysate) was loaded onto a column of Chelating Sepharose Fast Flow(Amersham Biosciences; Piscataway, N.J.) that had been charged withnickel ions. The lysate was captured after passing through the columnresin (break-through) and the column was washed extensively with IMACwash buffers A10, A15, and A20. The recombinant ME-2 bound to the resinwas eluted from the column with buffer containing imidazole. Samples ofthe lysate, breakthrough, wash, and elution were analyzed by SDS PAGEand Western blot as described above. The stained gel showed thecomplexity of the insect cell lysate, which resulted in a smear ofprotein for this, and the break-through samples. A substantial amount ofnon-binding protein contaminants were washed from the resin with theA10, A15, and A20 Column Wash buffers. Finally, a nice bandcorresponding to a 49 kD protein was present among the material elutedfrom the column with imidazol. Western blotting of these samples showedgood levels of the recombinant ME-2 protein in the lysate, and some alsoin the break-through showing that in this particular run the amount ofME-2 may have exceeded the binding capacity for the column. Perhaps atrace of ME-2 was present in the A10 Column Wash buffer, but strongersignals were detected in the A15 and A20 Column Wash buffers wash usedto remove bound impurities from the Sepharose. The Western showedintense anti-HisG antibody reactivity with the eluted and partiallypurified 49 kD ME-2 antigen.

FIG. 12 shows the isolation of the recombinant 6×-tagged EPP2 proteinusing immobilized metal affinity chromatography (IMAC). Recombinant EPP2protein was expressed in Sf9 insect cells and the cells were lysed indenaturing IMAC column binding buffer. The insect cell lysate was loadedonto a column of Chelating Sepharose Fast Flow (Amersham Biosciences;Piscataway, N.J.) that had been charged with nickel ions. The lysate wascaptured after passing through the column resin (break-through) and thecolumn was washed extensively with A10, A15, A20, A25, and A30 IMAC washbuffers. The recombinant EPP2 bound to the resin was eluted from thecolumn with buffer containing imidazole. Samples of the lysate,break-through, washes, and elution were analyzed by SDS PAGE and Westernblot as described above. The stained gel showed the complexity of theinsect cell lysate, which resulted in a smear of protein. In addition,the break-through and the A10 Column Wash samples contained asubstantial amount of material that did not bind to the column matrix.Very little protein contaminants were washed away with the A15, A20,A25, and A30 Column Wash buffers as visualized from the stained gel. Avery nice band corresponding to a 9 kD protein was present among thematerial eluted from the column with imidazol. Western blotting of thesesamples showed detectable levels of the recombinant EPP2 protein in thelysate. Little or no EPP2 was present in the break-through, A10, or A15samples showing that in this particular run the EPP2 bound to the columnpretty well. Perhaps a trace of EPP2 was detected in the in the A20,A25, and A30 Column Wash buffers used to remove bound impurities fromthe Sepharose. The Western showed intense anti-HisG antibody reactivitywith the eluted 9 kD EPP2 antigen.

FIG. 13 shows Western blot analysis of isolated recombinant ME-5protein, as well as the native ME-5 antigen present in RL95-2endometrial carcinoma cells. Cultured RL95-2 cells were lysed and asample of the soluble fraction electrophoresed in a 4% to 20% TrisGlycine SDS PAGE gel (Invitrogen; Carlsbad, Calif.). A sample ofrecombinant ME-5 isolated by IMAC from Sf9 insect cells was included onthe gel as a positive control for the anti-ME-5 antibody. Westernblotting was performed with the 2D1 anti-ME-5 monoclonal antibodyfollowed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody. Aclear band of reactivity was observed (right lane) among the RL95-2proteins that seemed to migrate with a molecular weight that wasslightly greater than the insect cell recombinant.

FIG. 14 is a Western blot showing ME-5 native antigen expression invarious human tissues. Tissue protein extracts in SDS PAGE sample buffer(protein medleys: BD Biosciences; San Diego, Calif.) were separated inSDS PAGE gels and Western blotting done as described in FIG. 13. Thenative ME-5 antigen seems to be ubiquitously present in all tissuesexamined, but it appears to be slightly more abundant in heart, liver,ovary and kidney extracts.

FIGS. 15A and 15B show representative line immunoblots illustrating theability of recombinant ME-5 to react with antibodies present in serumobtained from endometriosis patients, but not in normal control sera.Each strip contains immobilized antigens that were slotted onto themembrane at different concentrations. The protein concentrations forME-5 are 0.018, 0.036, 0.072, and 0.144 milligrams per milliliter(mg/ml). The optimal concentration for discrimination between patientsand controls was 0.036 mg/ml as designated by the arrow at the right ofthe line blot strips. One advantage of the line immunoblot assay is thatmany different proteins can be interrogated on a single strip, andadditional unrelated proteins are present on the strips that act asinternal controls. A reagent control (mouse anti-human IgG monoclonal)is included on each strip to act as a positive control. Each strip wasincubated with serum from a normal person (control) or from a patientwith confirmed endometriosis. Line blot patterns for a total of 11controls (A6, A7, A8, A9, A10, A14, A15, A16, A17, A18, A21) are shownin FIG. 15A. In addition, 23 endometriosis patients (DS01, DS02, DS03,DS04, DS05, DS06, DS07, DS08, DS10, DS11, DS12, DS13, DS27, DS28, DS29,DS30, DS31, DS32, DS33, DS34, DS36, DS38, DS39) are shown in FIG. 15B.The intensity of staining of each band is indicative of the reactivityof the tested serum with ME-5. In this selected lineblot panel, ME-5 ata concentration of 0.036 mg/ml detected 18 endometriosis patients aspositive (DS01, DS03, DS05, DS06, DS10, DS11, DS12, DS27, DS28, DS29,DS30, DS31, DS32, DS33, DS34, DS36, DS38, and DS39). In addition, 5endometriosis patients (DS02, DS04, DS07, DS08, and DS13) yieldedpatterns of reactivity that were a bit lower. Among the 11 normalcontrols, ME-5 clearly did not react with nine of them (A6, A7, A8, A10,A15, A16, A17, A18, A21). There may have been detectable signals seenfor two of the normal controls (A9, A14), but these were very lightrelative to the patterns seen with sera from the endometriosis patientsand are interpreted as negative.

FIGS. 16A and 16B show representative line immunoblots illustrating theability of recombinant ME-2 to react with antibodies present in serumobtained from endometriosis patients, but not in normal control sera.Each strip contains immobilized antigens that were slotted onto themembrane at different concentrations. The protein concentrations of ME-2applied to the strips are 0.009 (for endometriosis sera, only), 0.018,0.036, 0.072, and 0.144 (for control sera, only) milligrams permilliliter (mg/ml). The optimal concentration for discrimination betweenpatients and controls was set at 0.018 mg/ml as designated by the arrowat the right of the line blot strips. One advantage of the lineimmunoblot assay is that many different proteins can be interrogated ona single strip for reactivity with antibodies, and additional unrelatedproteins are present on the strips that act as internal controls. Areagent control (mouse anti-human IgG monoclonal) is included on eachstrip to capture human IgG and act as a positive control. Each strip wasincubated with serum from a normal person (control) or from a patientwith confirmed endometriosis. Line blot patterns for a total of 11controls (A01, A02, A03, A06, A08, A15, A20, A21, A22, A23, and A24) areshown in FIG. 16A. In addition, 21 endometriosis patients (DS10, DS11,DS12, DS13, DS14, DS17, DS19, DS20, DS21, DS22, DS24, DS25, DS26, DS27,DS28, DS29, DS30, DS31, DS32, DS33, and DS35) are shown in FIG. 16B. Theintensity of staining of each band is indicative of the reactivity ofthe tested serum with ME-2. In this selected lineblot panel, ME-2 at aconcentration of 0.018 mg/ml detected 15 endometriosis patients aspositive (DS012, DS17, DS19, DS20, DS21, DS22, DS24, DS25, DS26, DS27,DS28, DS30, DS31, DS33, and DS35). In addition, 6 endometriosis patients(DS10, DS11, DS13, DS14, DS29, and DS32) yielded patterns of reactivitythat were a bit lower. Among the 11 normal controls, ME-2 did not reactwith any of them at the 0.018 mg/ml cutoff applied to endometriosispatients.

FIGS. 17A and 17B show representative line immunoblots illustrating theability of recombinant EPP2 to react with antibodies present in serumobtained from endometriosis patients, but not in normal control sera.Each strip contains immobilized antigens that were slotted onto themembrane at different concentrations. The protein concentrations forEPP2 are 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, and 025 milligrams permilliliter. The optimal concentration for discrimination betweenpatients and controls was 0.05 mg/ml as designated by the arrow at theright of the line blot strips. One advantage of the line immunoblotassay is that many different proteins can be interrogated on a singlestrip, and additional unrelated proteins are present on the strips thatact as internal controls. A reagent control (mouse anti-human IgGmonoclonal) is included on each strip to capture human IgG in the sampleand act as a positive control. Each strip was incubated with serum froma normal person (control) or from a patient with confirmedendometriosis. Line blot patterns for a total of 11 controls (A01, A02,A03, A04, A05, A09, A13, A14, A16, A20, and A24) are shown in FIG. 17A.In addition, 39 endometriosis patients (DS06, DS12, DS24, DS05, BBI01,BBI02, BBI03, BBI04, BBI05, BBI06, BBI07, BBI08, BBI09, BBI10, BBI11,BBI12, BBI13, BBI14, BBI15, BBI16, BBI20, BBI21, BBI22, BBI23, BBI24,BBI25, BBI26, BBI27, BBI28, BBI30, BBI31, BBI32, BBI34, BBI35, BBI36,BBI37, BBI38, BBI39, and BBI40) are shown in FIG. 17B. The intensity ofstaining of each band is indicative of the reactivity of the testedserum with EPP2. In this selected lineblot panel, EPP2 at aconcentration of 0.05 mg/ml detected 33 endometriosis patients aspositive (DS06, DS12, DS24, DS05, BBI02, BBI03, BBI04, BBI06, BBI07,BBI08, BBI09, BBI10, BBI11, BBI12, BBI13, BBI15, BBI16, BBI20, BBI22,BBI23, BBI25, BBI26, BBI27, BBI28, BBI30, BBI31, BBI32, BBI34, BBI35,BBI37, BBI38, BBI39, and BBI40). In addition, 6 endometriosis patients(BBI01, BBI05, BBI14, BBI21, BBI24, and BBI36) yielded patterns ofreactivity that were much lower. Among the 11 normal controls, EPP2 didnot react strongly with any of them at the 0.05 mg/ml cut off.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring analogs thereof. Synthetic polypeptides canbe synthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. It is to beunderstood that the claims encompass conservative substitution. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methoinine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

“Allelic Variant” refers to any of two or more polymorphic forms of agene occupying the same genetic locus. Allelic variations arisenaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. “Allelic variants” also refer to cDNAs derived from mRNAtranscripts of genetic allelic variants, as well as the proteins encodedby them.

This invention provides methods for diagnosing endometriosis in asubject by detecting in a sample from the subject a diagnostic amount ofan antibody that specifically binds to ME-2, ME-5 or EPP2 polypeptide.Suitable patient samples include, without limitation, saliva, blood or ablood product (e.g., serum), peritoneal fluid, urine, menstrual fluid,vaginal secretion. The antibodies can be detected by any of the methodsfor detecting proteins described herein. However, sandwich type assaysare particularly useful. In one version, all antibodies are capturedonto a solid phase, for example using protein A, and antibodies specificfor ME-2, ME-5 or EPP2 are detected using a directly or indirectlylabeled ME-2, ME-5 or EPP2 or polypeptide fragment of it having anepitope of ME-2, ME-5 or EPP2. In another version of the assay, ME-2,ME-5 or EPP2 or an antigenic fragment of it can be used as the capturemolecule and captured antibodies can be detected.

ME-2, ME-5 or EPP2 that is shed into the peritoneal fluid of women withendometriosis is useful in methods of diagnosing endometriosis. Thesemethods include detecting ME-2, ME-5 or EPP2 in a biological sample of asubject. Suitable samples include, without limitation, saliva, blood ora blood product (e.g., serum), urine, menstrual fluid, vaginal secretionand, in particular, peritoneal fluid. ME-2, ME-5 or EPP2 can be detectedby any of the methods described herein. Any detection of ME-2, ME-5 orEPP2 above a normal range is a positive sign in the diagnosis ofendometriosis.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or sub-sequencesthat have at least 60%, 80%, 90%, 95% or 98% nucleotide or amino acidresidue identity, when compared and aligned for maximum correspondence,as measured using one of the following sequence comparison algorithms orby visual inspection. Preferably, the substantial identity exists over aregion of the sequences that is at least about 50 residues in length,more preferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues. In a most preferred embodiment, the sequences aresubstantially identical over the entire length of the coding regions.

The invention disclosed herein is the isolation of human cDNA moleculesthat encode three distinct endometrial proteins, and characterization ofthe corresponding antigens. These cDNA and the corresponding antigenshave been designated ME-5, ME-2, and EPP2 and the proteins expressedfrom them are the targets of autoantibodies present in the serum ofwomen who suffer from endometriosis. These features of the ME-5, ME-2,and EPP2 proteins makes them useful markers for diagnosis of endometrialdisease and this is shown in detail in the Examples below.

Example 1 Identification and Cloning of the ME-5, ME-2, AND EPP2 cDNAS

The endometriosis tissue cDNA library was generated using poly A⁺ RNAisolated from a deep embedded endometriosis tissue specimen donated byProfessor Philip Koninckx at the Catholic University of Leuven. TotalRNA was isolated from the tissue using Trizol reagent (BioradLaboratories; Hercules, Calif.), and poly A+ RNA was prepared byhybridization to oligo poly T coupled magnetic particles using acommercial kit (PolyATract; Promega; Madison, Wis.). Libraryconstruction was carried out using the Lambda ZAP® II vector systemfollowing instructions obtained from the supplier (Stratagene; SanDiego, Calif.). The initial ME-5 and ME-2 cDNA clones were identified byimmunoscreening using, as primary antibody, a single endometriosispatient serum specimen obtained from a woman diagnosed with milddisease. This serum was adsorbed of nonspecific anti-E. coli/lambdaphage antibodies by diluting the sera 1:50 in a commercial E. coli phagelysate (Stratagene; San Diego, Calif.) according to the protocolprovided by the supplier. In a separate series of experiments theinitial EPP2 cDNA clone was identified in similar immunoscreeningprotocol except that, as primary antibody, a pool of ten endometriosispatient serum specimens was used. The sera in this pool were from womenwith various stages of endometrial disease. Again the serum was adsorbedof nonspecific anti-E. coli/lambda phage antibodies by dilution with acommercial E. coli phage lysate (Stratagene; San Diego, Calif.) asdescribed above. The second antibody for all screening experiments was¹²⁵-labeled monoclonal antibody reactive with human immunoglobulin.Negative control human serum was used to screen the clones in parallelto verify the reactivity. Immunoreactive clones were plaque-purifiedthree times and rescued by in vivo excision into the pBluescript® SK(−)phagemid vector using methods supplied by the manufacturer (Stratagene;San Diego, Calif.).

Example 2 Characterization of ME-5, ME-2, and EPP2 cDNA and Protein

Sequence analysis of both strands of each of the original isolated ME-5,ME-2, and EPP2 clones was performed upon an ABI Biosystems 373 DNASequencer (PE Applied Biosystems; Foster City, Calif.). The nucleic acidsequences so generated were analyzed using Bionet software to identifynucleic acid and protein characteristics and for homology comparisonswith nucleic acid and protein sequences present in the database.

The ME-5 cDNA sequence is presented in FIG. 1A (SEQ ID NO:1) and it is1,279 base pairs in size excluding the poly dA track. A 5′ noncodingsequence of 112 base pairs was identified just upstream of the suspectedATG start codon. There is a 3′ non coding sequence of 254 base pairsdown stream of the TGA stop codon and this is followed by a stretch ofdA residues that would correspond to the poly A tail at the 3′ end ofthe mRNA. Both the start and stop codon are highlighted in bold type inFIGS. 1A and 1B. The ME-5 coding sequence is shown in FIG. 1B (SEQ IDNO:2) as predicted from the entire isolated cDNA sequence (FIG. 1A). Thecoding region is 912 base pairs in size, including the start and stopcodons. The cDNA codes for a predicted protein of 303 amino acids shownin FIG. 1C (SEQ ID NO:3) and the calculated molecular weight was about35,000 Daltons. The translation product is slightly acidic with acalculated isoelectric value of 5.7.

Computer-assisted database searches (National Center for BiotechnologyInformation [NCBI] Basic Local Alignment Search Tool [BLAST]) was usedto perform homology comparisons with sequences contained within theGenBank nucleic acid database. It was discovered that two otherlaboratories working on different projects independently isolated anessentially identical cDNA molecule.

First Scanlan and coworkers isolated the identical 1-NY-CO-7 cDNA usinga process described in a paper: “Characterization of human colon cancerantigens recognized by autologous antibodies” published by Scanlan etal. [Int. J. Cancer 76, 652-658 (1998)]. The approach used by theseindividuals was similar to that employed for discovery of the ME-5 cDNAin that these investigators screened colorectal cancer cDNA librarieswith serum from colorectal cancer patients. Comparing the ME-5 sequencewith that of 1-NY-CO-7 revealed a substantial number of differencesbetween the two. First, in the manuscript the 1-NY-CO-7 mRNA sequencewas reported to be 1.22 kb perhaps slightly smaller than the ME-5sequence of this invention. Second, the 1-NY-CO-7 protein was reportedto be 356 amino acids on size which is considerably larger than thepredicted ME-5 protein. Finally, there were three base mismatches in thecarboxy terminal portion of the two sequences and two of these resultedin amino acid changes. The nucleotide changes were at nucleotide 807(C->G [occurs in 3′ position of codon with no amino acidchange->proline]), 814 (C->G [arginine->glycine]), and 838 (C->T[leucine->phenylalanine]) relative to the ME-5 coding domain. Theauthors commented that the 1-NY-CO-7 sequence was novel (little or nohomologies with DNA sequences listed in the Gen Bank/EMBO data baseswith the exception of expressed sequence tags), and the protein ashaving tetratricopeptide repeats (TPR, see below). The 1-NY-CO-7sequence did not appear among those colon-specific sequences that werecharacterized in the paper, rather it was a direct submission to GenBankwithout further characterization of the nucleic acid or protein. Whenthe 1-NY-CO-7 GenBank sequences were compared to that of ME-5 they wereidentical except for the three nucleotide mismatches described above.

Second, Ballinger and coworkers identified the identical Carboxyterminus of Hsp70-interacting protein (CHIP) using a drasticallydifferent process described in a paper published: “Identification ofCHIP, a novel tetratricopeptide repeat-containing protein that interactswith heat shock proteins and negatively reglates chaperone functions”Ballinger et al. [Mol. Cell. Biol. 19, 4535-4545 (1999)]. In this paperthe authors were interested in isolating novel tetratricopeptiderepeat-containing proteins. The CHIP sequence was identified byscreening a cardiac cDNA library with the cDNA sequence for the humanCyP-40 protein at different stringency's. A low stringency hybridization(42° C.) yielded 12 clones that did not hybridize at higher stringency(55° C.). Characterization of the clones revealed 8 of them correspondedto human CyP-40, and 4 clones encoded CHIP that was a sequence with nohomology to known genes. Characterization of CHIP revealed that itinteracts with both Hsc70 and Hsp70 by binding to the carboxy terminusof these proteins through sequences within the amino terminus of CHIP.Interestingly recombinant CHIP inhibited the Hsp40-stimulated ATPaseactivity of Hsc70 and Hsp70 suggesting that it regulated the forwardreaction of the substrate-binding cycle.

Both of these sequences have near perfect homology with the ME-5 nucleicacid and protein sequences of this invention. However, the anticipatedusefulness of ME-5 in the diagnosis of endometriosis was notcontemplated by the aforementioned papers.

The ME-2 cDNA sequence is presented in FIG. 2A (SEQ ID NO:4) and it is1,332 base pairs in size excluding the poly dA track. A 5′ noncodingsequence of 54 base pairs was identified just upstream of the suspectedATG start codon. There is a 3′ non coding sequence of 95 base pairs downstream of the TAG stop codon and this is followed by a stretch of dAresidues that would correspond to the poly A tail at the 3′ end of themRNA. Both the start and stop codon are highlighted in bold type inFIGS. 2A and 2B. The ME-2 coding sequence is shown in FIG. 2B (SEQ IDNO:5) as predicted from the entire isolated cDNA sequence (FIG. 2A). Thecoding region is 1182 base pairs in size, including the start and stopcodons. The cDNA codes for a predicted protein of 393 amino acids shownin FIG. 2C (SEQ ID NO:6) and the calculated molecular weight was about45,000 Daltons. The translation product is slightly acidic with acalculated isoelectric value of 8.8.

Computer-assisted database searches (National Center for BiotechnologyInformation [NCBI] Basic Local Alignment Search Tool [BLAST]) was usedto perform homology comparisons with the ME-2 cDNA with sequencescontained within the GenBank nucleic acid database. It was discoveredthat while there are several submissions by groups involved withanalysis of the human genome sequence all documents are directsubmissions. Moreover, none of these submissions have been published inthe scientific literature, and all refer to “unknown protein” or“hypothetical protein” or “unnamed protein product” and not to a definedproduct or function. These can be found in accession numbersGI:12652526, GI:22761484, and GI:24431994 for example. Therefore eventhough sequences corresponding to the ME-2 cDNA and protein are presentin the public domain, the nature is not known and the involvement of theprotein in endometriosis is certainly not-anticipated by this publicinformation. Consequently, the ME-2 cDNA and protein sequences areunique and exclusively implicated in the human disease of endometriosisby the disclosures contained in this invention.

The EPP2 cDNA sequence is presented in FIG. 3A (SEQ ID NO:7) and it is868 base pairs in size excluding the poly dA track. A 5′ noncodingsequence of 45 base pairs was identified just upstream of the suspectedATG start codon. There is a 3′ non coding sequence of 522 base pairsdown stream of the TAA stop codon and this is followed by a stretch ofdA residues that would correspond to the poly A tail at the 3′ end ofthe mRNA. Both the start and stop codon are highlighted in bold type inFIGS. 3A and 3B. The EPP2 coding sequence is shown in FIG. 3B (SEQ IDNO:8) as predicted from the entire isolated cDNA sequence (FIG. 3A). Thecoding region is 300 base pairs in size, including the start and stopcodons. The cDNA codes for a predicted protein of 99 amino acids shownin FIG. 3C (SEQ ID NO:9) and the calculated molecular weight wasapproximately 9300 Daltons. Interestingly 18 of the amino acids arearginine residues therefore the translation product is very basic with acalculated isoelectric value of 12.5.

Computer-assisted database searches (National Center for BiotechnologyInformation [NCBI] Basic Local Alignment Search Tool [BLAST]) was usedto perform homology comparisons with the EPP2 cDNA with sequencescontained within the GenBank nucleic acid database. In the fashiondescribed above for ME-2, it was discovered that EPP2 was alsorepresented by several direct submissions from groups involved withanalysis of the human genome sequence. Moreover, as described above,none of these submissions have been published in the scientificliterature, and all refer to “unknown protein” or “hypothetical protein”or “unnamed protein product” and not to a defined product or function.These can be found in accession numbers GI:12652993, GI:24308450, andGI:20892293 for example. Therefore even though sequences correspondingto the EPP2 cDNA and protein are present in the public domain the natureis not known and the involvement of the protein in endometriosis iscertainly not anticipated by this public information. Consequently, theEPP2 cDNA and protein sequences are unique and exclusively implicated inthe human disease of endometriosis by the disclosures contained in thisinvention.

Example 3 Northern Blotting with Radiolabeled ME-5, ME-2, and EPP2Probes: mRNA Character and Expression Pattern

Gene expression profile of ME-5 from normal human tissues was done byperforming Northern blot analysis with a commercial Multiple TissueNorthern Blot (BD Biosciences; San Diego, Calif.), the results of whichare presented in FIG. 4. The commercial Northern blot contained RNA fromthe following tissues: spleen, thymus, prostate, testis, uterus, smallintestine, colon (no mucosa), and peripheral blood leukocyte. The entire912 base pair coding sequence was isolated by electrophoresis in a lowmelting agarose gel, and labeled with ³²P by random priming. The³²P-labeled ME-5 probe was used for hybridization to the Northern blotusing the procedure supplied by the manufacturer. After washing the blotwas exposed to X-ray film. Upon development of the film a band at about1.4 kb on the Northern blot corresponds to the ME-5 transcript of theexpected size (FIG. 4). The transcript can be seen in all tissues and isparticularly abundant in prostate, testis and uterus tissues.

Gene expression profile of ME-2 from normal human tissues was done byperforming Northern blot analysis with a commercial Multiple TissueNorthern Blot (BD Biosciences; San Diego, Calif.), the results of whichare presented in FIG. 5. The commercial Northern blot contained RNA fromthe following tissues: spleen, thymus, prostate, testis, uterus, smallintestine, colon (no mucosa), and peripheral blood leukocyte. The entire1182 base pair coding sequence was isolated by electrophoresis in a lowmelting agarose gel, and labeled with ³²P by random priming. The³²P-labeled ME-2 probe was used for hybridization to the Northern blotusing the procedure supplied by the manufacturer. After washing the blotwas exposed to X-ray film. Upon development of the film a band at about2.0 kb on the Northern blot corresponds to the ME-2 transcripthybridizing to the labeled probe (FIG. 5). The transcript can be seen inall tissues and is particularly abundant in prostate and testis. Inaddition, good levels of hybridization were observed amount the RNAsexpressed in spleen, uterus, small intestine, colon, and peripheralblood lymphocyte tissues. Interestingly, despite the pattern observedwith the peripheral blood lymphocytes, relatively little signal could bedetected in thymus tissue.

Gene expression profile of EPP2 from normal human tissues was done byperforming Northern blot analysis with a commercial Multiple TissueNorthern Blot (BD Biosciences; San Diego, Calif.), the results of whichare presented in FIG. 6. The commercial Northern blot contained RNA fromthe following tissues: spleen, thymus, prostate, testis, uterus, smallintestine, colon (no mucosa), and peripheral blood leukocyte. The entire300 base pair EPP2 coding sequence was isolated by electrophoresis in alow melting agarose gel, and labeled with ³²P by random priming. The³²P-labeled EPP2 probe was used for hybridization to the Northern blotusing the procedure supplied by the manufacturer. After washing the blotwas exposed to X-ray film. Upon development of the film a band at about1.0 kb on the Northern blot corresponds to the EPP2 transcripthybridizing to the labeled probe (FIG. 6). The transcript can be seen inall tissues and is most abundant in prostate, testis, colon, andperipheral blood lymphocyte tissues. In addition, the transcript ispresent but the relative levels of hybridization are lower among theRNAs expressed in spleen, thymus, uterus, and small intestine tissues.

Example 4 Expression of Recombinant ME-5, ME-2, and EPP2 Proteins in anInsect Cell Host

The ME-5 antigen was cloned for expression as a 6× histidine-taggedfusion protein in insect cells. The sequence of the ME-5 cDNA insert wasgenerated by PCR amplification using specific primers that flanked the912 by coding region. Unique sites for the Bam HI and Eco RI restrictionenzymes were incorporated into the primers to maintain the ME-5 readingframe with the vector sequences. The PCR amplicons were digested withthe Barn HI and Eco RI restriction enzymes (Stratagene; San Diego,Calif.) and purified by agarose gel electrophoresis. The insect celltransfer vector Blue Bac His2a (Stratagene; San Diego, Calif.) was alsodigested with the restriction enzymes Barn HI and Eco RI and treatedwith calf intestine alkaline phosphatase. The ME-5 cDNA insert wasligated with the vector and competent bacteria transformed. Individualisolated clones were grown, plasmid DNA isolated, and digested with therestriction enzymes Barn HI and Eco RI. Clones producing a band of about900 by in addition to the linear vector were chosen. Several candidateswere further characterized by DNA sequence analysis to verify that nochanges occurred during the process of PCR amplification and cloning.One clone was confirmed to have no mutations and this was used fordevelopment of recombinant baculovirus vectors. Recombinantbaculoviruses were generated by cotransfection of Sf9 insect cells withbaculovirus DNA and the ME-5 transfer vector. The baculoviruses wereisolated by plaque purification and used to evaluate expression patternsin pilot cultures. The recombinant baculovirus virus and pilot scalecultures were evaluated for expression patterns. One recombinantbaculovirus clone was identified which expressed an antigen ofapproximately 38 kD, which was detected in both the soluble and theinsoluble fraction of the insect cell lysates. The clone was expandedinto large-scale virus stocks for expression of recombinant ME-5protein. This was used to infect a large-scale culture of Sf9 insectcells. The pattern of expression is illustrated in FIG. 7, and is bestvisualized by the Western blot analysis (FIG. 7). The presence ofrecombinant ME-5 was confirmed with a commercial anti-HisG monoclonalantibody (Invitrogen; Carlsbad, Calif.) followed by an ¹²⁵I-labeledrabbit anti-mouse IgG secondary antibody. This confirms the presence ofa 6× histidine-tagged protein of approximately 38 kD which is themolecular weight expected for ME-5. The recombinant was detected in boththe soluble and the insoluble fraction of the insect cell lysates, butslightly more antigen seems to be localized in the soluble fraction. Inaddition some antigen was present in the PBS used to wash the infectedcells prior to the lysis.

The ME-2 antigen was cloned for expression as a 6× histidine-taggedfusion protein in insect cells as described above for the ME-5 activity.The sequence of the ME-2 cDNA insert was generated by PCR amplificationusing specific primers that flanked the 1182 by coding region. Uniquesites for the Bam HI and Eco RI restriction enzymes were incorporatedinto the primers to maintain the ME-2 reading frame with the vectorsequences. The PCR amplicons were digested with the Bam HI and Eco RIrestriction enzymes (Stratagene; San Diego, Calif.) and purified byagarose gel electrophoresis. The insect cell transfer vector Blue BacHis2a (Stratagene; San Diego, Calif.) was also digested with therestriction enzymes Barn HI and Eco RI and treated with calf intestinealkaline phosphatase. The ME-2 cDNA insert was ligated with the vectorand competent bacteria transformed. Individual isolated clones weregrown, plasmid DNA isolated, and digested with the restriction enzymesBarn HI and Eco RI. Clones producing a band of about 1100 by in additionto the linear vector were chosen. Several candidates were furthercharacterized by DNA sequence analysis to verify that no changesoccurred during the process of PCR amplification and cloning. One clonewas confirmed to have no mutations and this was used for development ofrecombinant baculovirus vectors. Recombinant baculoviruses weregenerated by cotransfection of Sf9 insect cells with baculovirus DNA andthe ME-2 transfer vector. The baculoviruses were isolated by plaquepurification and used to evaluate expression patterns in pilot cultures.The recombinant baculovirus virus and pilot scale cultures wereevaluated for expression patterns. One recombinant baculovirus clone wasidentified which expressed an antigen of approximately 49 kD, which wasdetected in both the soluble and the insoluble fraction of the insectcell lysates. The clone was expanded into large-scale virus stocks forexpression of recombinant ME-2 protein. This was used to infect alarge-scale culture of Sf9 insect cells. The pattern of expression isillustrated in FIG. 8, and is best visualized by the Western blotanalysis (FIG. 8). The presence of recombinant ME-2 was confirmed with acommercial anti-HisG monoclonal antibody (Invitrogen; Carlsbad, Calif.)followed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody.This confirms the presence of a 6× histidine-tagged protein ofapproximately 49 kD which is the molecular weight expected for ME-2. Therecombinant was detected in both the soluble and the insoluble fractionof the insect cell lysates, but slightly more antigen seems to belocalized in the soluble fraction. In addition some antigen was presentin the PBS used to wash the infected cells prior to the lysis.

The EPP2 antigen was cloned for expression as a 6× histidine-taggedfusion protein in insect cells as described above. The sequence of theEPP2 cDNA insert was generated by PCR amplification using specificprimers that flanked the 300 by coding region. Unique sites for the BarnHI and Eco RI restriction enzymes were incorporated into the primers tomaintain the EPP2 reading frame with the vector sequences. The PCRamplicons were digested with the Bam HI and Eco RI restriction enzymes(Stratagene; San Diego, Calif.) and purified by agarose gelelectrophoresis. The insect cell transfer vector Blue Bac His2a(Stratagene; San Diego, Calif.) was also digested with the restrictionenzymes Bam HI and Eco RI and treated with calf intestine alkalinephosphatase. The EPP2 cDNA insert was ligated with the vector andcompetent bacteria transformed. Individual isolated clones were grown,plasmid DNA isolated, and digested with the restriction enzymes Bam HIand Eco RI. Clones producing a band of about 300 by in addition to thelinear vector were chosen. Several candidates were further characterizedby DNA sequence analysis to verify that no changes occurred during theprocess of PCR amplification and cloning. One clone was confirmed tohave no mutations and this was used for development of recombinantbaculovirus vectors. Recombinant baculoviruses were generated bycotransfection of Sf9 insect cells with baculovirus DNA and the EPP2transfer vector. The baculoviruses were isolated by plaque purificationand used to evaluate expression patterns in pilot cultures. Therecombinant baculovirus virus and pilot scale cultures were evaluatedfor expression patterns. One recombinant baculovirus clone wasidentified which expressed an antigen of approximately 9 kD, which wasdetected in both the soluble and the insoluble fraction of the insectcell lysates. The clone was expanded into large-scale virus stocks forexpression of recombinant EPP2 protein. This was used to infect alarge-scale culture of Sf9 insect cells. The pattern of expression isillustrated in FIG. 9, and is best visualized by the Western blotanalysis (FIG. 9). The presence of recombinant EPP2 was confirmed with acommercial anti-HisG monoclonal antibody (Invitrogen; Carlsbad, Calif.)followed by an ¹²⁵I-labeled rabbit anti-mouse IgG secondary antibody.This confirms the presence of a 6× histidine-tagged protein ofapproximately 9 kD which is the molecular weight expected for EPP2. Therecombinant was detected in both the soluble and the insoluble fractionof the insect cell lysates, but slightly more antigen seems to belocalized in the soluble fraction. In contrast to the patterns seen withthe ME-5 and ME-2 protein expression, no EPP2 antigen was present in thePBS used to wash the infected cells prior to the lysis.

Example 5 Purification of Recombinant ME-5, ME-2, and EPP2 Protein fromInsect Cells

Additional studies of the ME-5, ME-2, and EPP2 antigens requiresubstantial amounts of isolated protein. Specifically, these are neededfor evaluating the reactivity of the ME-5, ME-2, and EPP2 proteins withendometriosis patient serum specimens to establish clinical relevance.The recombinant ME-5, ME-2, and EPP2 antigens were isolated from thesoluble fraction or the whole cell lysate by immobilized metal affinitychromatography (IMAC).

Recombinant ME-5 antigen was isolated from the soluble fraction of theinsect cell lysate. Briefly, ME-5 recombinant baculoviruns-infectedinsect cells were harvested after three days of infection bycentrifugation. The cells were washed twice with PBS and the cell pelletfrozen for one hour at −70° C. After thawing the cell pellet wassuspended in binding buffer (500 mM NaCl, 20 mM Tris-HCl, pH 8.0)supplemented with protease inhibitor cocktail for mammalian tissues(Sigma; St. Louis, Mo.). The lysate was sonicated, and centrifuged at18,000 rpm, 4° C. for 20 minutes to separate the soluble and insolublefractions. The soluble fraction was dialyzed against binding buffer, andcentrifuged at 18,000 rpm, 4° C. to remove impurities that might affectthe performance of the column. Nickel-charged chelating Sepharose resin(Amersham Biosciences; Piscataway, N.J.) was equilibrated twice with 2×column volume of binding buffer. The resin was incubated with the ME-5insect cell lysate for 20 minutes on a rocker at room temperature. Theresin/lysate mixture is loaded on a column and washed with 40 columnvolumes of A20 Column Wash buffer (20 mM imidazole; 500 mM NaCl; 20 mMTris-HCl, pH 8.0). Bound ME-5 protein was eluted from the column withelution buffer (500 mM imidazole; 500 mM NaCl; 20 mM Tris-HCl, pH 7.5).Protease inhibitor cocktail was added to the pooled elution fractionsand protein concentration measured by BCA assay (Pierce; Rockford, Ill.)using a BSA standard curve. The eluted protein samples are analyzed uponSDS PAGE followed by staining with Coomassie blue or transfer tonitrocellulose and Western blotting as shown in FIG. 10. Such isolatedME-5 protein preparations are divided into aliquots and stored at −20°C. with 30% glycerol.

Recombinant ME-2 antigen was also isolated from the soluble fraction ofthe insect cell lysate. Briefly, ME-2 recombinant baculovirus-infectedinsect cells were harvested after three days of infection bycentrifugation. The cells were washed with PBS and the cells lysed asdescribed above. After dialysis the soluble fraction was allowed to bindto nickel-charged chelating Sepharose resin (Amersham Biosciences;Piscataway, N.J.) for 20 minutes at room temperature. The resin/lysatemixture is loaded on a column and washed sequentially with denaturingbinding buffer A10 (10 mM imidazole, 1 M NaCl, 20 mM Tris-HCl, pH 8.0,10% glycerol, 6 M Urea), A15 (buffer A10 containing 15 mM imidazole),and A20 (buffer A10 with 20 mM imidazole). Bound ME-2 protein was elutedfrom the column with elution buffer (500 mM imidazole; 500 mM NaCl; 20mM Tris-HCl, pH 7.5). Protease inhibitor cocktail was added to thepooled elution fractions and protein concentration measured by BCA assay(Pierce; Rockford, Ill.) using a BSA standard curve. The eluted proteinsamples are analyzed upon SDS PAGE followed by staining with Coomassieblue or transfer to nitrocellulose and Western blotting as shown in FIG.11. Such isolated ME-2 protein preparations are divided into aliquotsand stored at −20° C. with 30% glycerol.

Recombinant EPP2 antigen was isolated from the whole insect cell lysateas follows. The EPP2 recombinant baculovirus-infected insect cells wereharvested after three days of infection by centrifugation. The cellswere washed twice with PBS and the cell pellet frozen for one hour at−70° C. After thawing the cell pellet was suspended in denaturingbinding buffer (750 mM NaCl; 20 mM Tris-HCl, pH 8.0; 10% glycerol; 6 Mguanidine HCl) supplemented with protease inhibitor cocktail formammalian tissues (Sigma; St. Louis, Mo.). The lysate was sonicated, andcentrifuged to separate the soluble and insoluble fractions. The solublefraction was allowed to bind to nickel-charged chelating Sepharose resin(Amersham Biosciences; Piscataway, N.J.) for 20 minutes at roomtemperature. The resin is loaded on a column and washed sequentiallywith denaturing binding buffer A10 (10 mM imidazole, 1 M NaCl, 20 mMTris-HCl, pH 8.0, 10. % glycerol, 6 M Urea), A15 (buffer A10 with 15 mMimidazole), A20 (buffer A10 with 20 mM imidazole), A25 (buffer A10 with25 mM imidazole), and A30 (same as buffer A10 but with 30 mM imidazole).The isolated EPP2 protein is eluted with denaturing elution buffer (250mM imidazole, 1 M NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 6 M Urea).Protease inhibitor cocktail was added to the pooled elution fractionsand EPP2 protein is dialyzed against 0.2 M bicarbonate buffer with 0.5 MNaCl and cysteine/cystine to remove the urea. After dialysis the samplesare concentrated if needed on Aquacide and the protein concentrationmeasured by BCA assay (Pierce; Rockford, Ill.) using a BSA standardcurve. The isolated EPP2 protein samples are analyzed upon SDS PAGEfollowed by Coomassie and Western blotting as shown in FIG. 12. Proteinsamples are stored at −20° C. with 30% glycerol. Such isolated EPP2protein preparations are divided into aliquots and stored at −20° C.with 30% glycerol.

Example 6 Antibody Development

Monoclonal antibodies to the ME-5 protein were produced using standardmethods (G. Galfre et al. [1977] Nature 266:550) with modifications(V.T. Oi and L. A. Herzenberg [1980] In B. B. Mishell and S. M. Shiigi[eds.] Selected Method in Cellular Immunology [San Francisco: W.H.Freeman]). Such monocronal antibody reagents are valuable for additionalstudies of the ME-5 protein character, and to assist in development ofimmunoassays for determining the clinical significance of the protein inendometriosis patients. Mice (BALB/c) were immunized with isolatedrecombinant ME-5 antigen and the antibody response to the antigenmonitored in these animals by ELISA and Western blot techniques with theanimal's serum. When the antibody response was significant the animalswere boosted with another immunization with the ME-5 antigen. Three dayslater the spleen was removed from an animal and the immune-cellsisolated from the organ. The isolated spleen cells were fused with theimmunoglobulin non-producing Sp2/0 mouse myeloma cell line (M. Shulmanet al. [1978] Nature 276:269). The resulting hybridoma cells wereselected in culture medium containing HAT reagents. Candidate hybridomacells were cloned a minimum of two times by limiting dilution and theclones screened by ELISA using isolated ME-5 antigen. One hybridoma cellline designated 2D1 was found to react particularly well with theisolated ME-5 antigen and this was selected for additional experiments.

Example 7 Identification of Native ME-5 Antigen and Tissue Distribution

Initially the 2D1 monoclonal was used in Western blotting experimentsupon protein extracts obtained from cultured RL95-2 endometrialcarcinoma cells. Cultured RL95-2 cells were lysed by sonication, andinsoluble debris removed by centrifugation. A portion of the solublefraction was analyzed on SDS PAGE gels adjacent to a sample of isolatedrecombinant ME-5 protein. FIG. 13 shows the pattern of Western blotanalysis obtained with the anti-ME-5 2D1 monoclonal antibody. Themonoclonal reacted well with the isolated recombinant antigen and 2D1reactivity was seen at an estimated molecular weight of 38 kD. In theRL95-2 protein extract there was a clear band of reactivity with thenatural ME-5 protein that appeared to be slightly larger than theisolated recombinant.

These studies of the natural ME-5 antigen were expanded to includetissue extracts obtained from various human organs. In these Westernblotting experiments various tissues were studied including humanspleen, brain, lung, heart, liver, ovary, placenta, testis, skeletalmuscle, and kidney. Samples of commercially available human tissueprotein extracts (Protein Medleys: BD Biosciences; San Diego, Calif.)were separated by electrophoresis on SDS PAGE gels using instructionsprovided by the manufacturer. The protein extracts were evaluated byWestern blot using the anti-ME-5 2D1 monoclonal as primary antibody andthe immune complexes were detected with a ¹²⁵I-labeled anti-mouseantibody. The results are shown in FIG. 14 and there was a strong bandof reactivity with the isolated recombinant ME-5 protein included as acontrol. In addition, natural ME-5 protein seems to be present in nearlyall of the tissues examined, and appropriately the protein seems to bepresent at the highest concentrations in ovary tissue. The presence ofthe ME-5 protein in other reproductive tissue is taken to imply that theantigen may naturally be expressed at high levels in this class oforgans. It is therefore likely that ME-5 may have a major role inregulation of functions occurring in the reproductive system.Consequently ME-5 would be more likely to be the target ofanti-endometrial antibodies generated during endometriosis. Good levelsof expression were also registered in heart, liver, and kidney tissueextracts. This is also encouraging because the CHIP protein discussed inExample 2 was isolated from a cardiac cDNA library. Somewhat lower, butdetectable levels of natural ME-5 were found in most of the remainingtissue extracts.

Example 8 Clinical Findings: Reactivity of the ME-5, ME-2, and EPP2Antigens with Endometriosis Patient Sera

The clinical significance of the ME-5, ME-2 and EPP2 proteins wereevaluated using line immunoblotting studies to measure reactivity withantibodies present in the serum of endometriosis patients. These lineblotting experiments were designed to identify IgG antibodies in humanserum reactive with the recombinant proteins. Briefly, the line blotutilizes the ME-5, ME-2, and EPP2 recombinant protein antigens which areimmobilized on a nitrocellulose membrane in a discrete location and inthe form of a line spanning the surface. In addition, a reagent controlline is included to verify that the specific assay conditions have beenfollowed. After cutting the membrane into a number of individual stripsthese are incubated with individual patient serum, and patient IgG bindsto antigens immobilized in the discrete lines. Immune complexes arevisualized by incubating the strips with enzyme-labeled anti-human IgGantibodies and a subsequent substrate reaction. When evaluating theseexperiments, the strips treated with control sera were compared to thestrips incubated with endometriosis patient sera to facilitate analysisof the intensity of staining of each band. The patient serum specimenshowing reactivity with the ME-5, ME-2, and EPP2 antigens is consideredpositive if the intensity of the signal is stronger than that obtainedwith the protein on the control patient strips.

Some representative lineblot strips containing ME-5 antigen treated withnormal control patient serum are shown in FIG. 15A. In contrast, thepattern of reactivity of representative endometriosis patients withsimilar strips is shown in FIG. 15B. Sera from endometriosis patientsconsistently react much more strongly with the ME-5 protein whencompared to control sera (compare patterns of 15A and 15B). Generally aparticular concentration of the ME-5 recombinant is signaled out whichoffers the best discrimination of reactivity for antibodies inendometriosis patients relative to controls. In these experiments, andothers to be summarized later, this concentration was 0.036 milligramsof ME-5 per milliliter. At this value, few if any control patientsreact, but reactivity of the endometriosis patients was substantial.

In this experiment a total of 47 endometriosis patients sera (DiagnosticSupport; Boston, Mass.) were evaluated along with 24 negative controls.Some representative data are shown in the figures discussed above, andthe reactivity of all endometriosis patient sera with the recombinantME-5 antigen is summarized in Table 1. The control reactivity ispresented in Table 2. A total of 27 of the endometriosis patients werestrongly positive in this experiment. Moreover, most of those patientsera that seemed to be below the 0.036 milligrams of ME-5 per millilitercut off definitely reacted better with higher concentrations of antigencompared to the negative control sera as noted above. None of thecontrol specimens had had very much antibody reactivity to the ME-5antigen as measured by line blot. Therefore, the marker ME-5 reactedwith at least 57% of the endometriosis patient sera evaluated in thisexperiment, and overall the pattern of reactivity of sera fromendometriosis patients was considerably stronger with the ME-5 proteinwhen compared to the patterns observed with control sera.

Some representative lineblot strips containing the ME-2 antigen andtreated with normal control patient serum are shown in FIG. 16A. Incontrast, the pattern of reactivity of representative endometriosispatients with similar strips is shown in FIG. 16B. Sera fromendometriosis patients consistently react much more strongly with theME-2 protein when compared to control sera (compare patterns of 16A and16B). Generally a particular concentration of the ME-2 recombinant issignaled out which offers the best discrimination of reactivity forantibodies in endometriosis patients relative to controls. In theseexperiments, and others to be summarized later, this concentration was0.018 milligrams of ME-2 per milliliter. At this value, few if anycontrol patients react, but reactivity of the endometriosis patients wassubstantial.

In this experiment a total of 47 endometriosis patients sera (DiagnosticSupport; Boston, Mass.) were evaluated for reactivity with the ME-2antigen along with 24 negative controls. Some representative data areshown in the figures discussed above, and the reactivity of all theendometriosis patient sera with the recombinant ME-2 antigen issummarized in Table 3. The control patient reactivity is presented inTable 4. A total of 25 of the endometriosis patients were stronglypositive in this experiment. Moreover, most of those patient sera thatseemed to be below the 0.018 milligrams of ME-2 per milliliter cut offdefinitely reacted better with higher concentrations of antigen comparedto the negative control sera as noted above. None of the controlspecimens had very much antibody reactivity to the ME-2 antigen asmeasured by line blot. Therefore, the marker ME-2 reacted with at least53% of the endometriosis patient sera evaluated in this experiment, andoverall the pattern of reactivity of sera from endometriosis patientswas considerably stronger with the ME-2 protein when compared to thepatterns observed with control sera. This is further exemplified by thefact that a two-fold higher concentration (0.144 mg/ml) of the ME-2antigen was evaluated for reactivity with control sera, and the signalintensity observed for this was considerably lower than that generatedwith endometriosis sera at lower protein levels.

As noted for the activities described above, the patient serum specimenshowing reactivity with EPP2 antigen is considered positive if theintensity of the signal is stronger than that obtained with the proteinon the control patient strips. Also noted above is that when evaluatingthese experiments, the strips treated with control sera were compared tothe strips incubated with endometriosis patient sera to facilitateanalysis of the intensity of staining of each band. Some representativelineblot strips showing the reactivity of recombinant EPP2 with normalcontrol patient serum are shown in FIG. 17A. In contrast, the pattern ofreactivity of representative endometriosis patients with similar stripsis shown in FIG. 17B. Generally sera from endometriosis patients reactmuch more strongly with the EPP2 protein when compared to control sera(compare patterns of 17A and 17B). Generally a particular concentrationof the EPP2 recombinant is signaled out which offers the bestdiscrimination of reactivity for antibodies in endometriosis patientsrelative to controls. In these experiments, and others to be summarizedlater, this concentration was 0.05 milligrams of EPP2 per milliliter. Atthis value, few if any control patients react, but reactivity of theendometriosis patients was substantial.

In this experiment a total of 90 endometriosis patients sera (DiagnosticSupport; Boston, Mass. and Boston Biomedica; Boston) were evaluatedalong with 24 negative controls. Some representative data are shown inthe figures discussed above, and the reactivity of all endometriosispatient sera with the recombinant EPP2 antigen is summarized in Table 5.The control patient reactivity presented in Table 6. A total of 55 ofthe endometriosis patients were strongly positive in this experiment.Moreover, most of those patient sera that seemed to be below the 0.05milligrams of EPP2 per milliliter cut off definitely reacted better withhigher concentrations of antigen compared to the negative control seraas noted above. None of the control specimens had very much antibodyreactivity to the EPP2 antigen as measured by line blot. Therefore, themarker EPP2 reacted with at least 61% of the endometriosis patient seraevaluated in this experiment, and overall the pattern of reactivity ofsera from endometriosis patients was considerably stronger with the EPP2protein when compared to the patterns observed with control sera.

Overall the pattern of reactivity for the individual ME-5, ME-2, andEPP2 antigens was between 53% and 61% and this seems sufficient to beuseful as a diagnostic marker for endometriosis. However, uponexamination of the results summarized in Tables 1, 3, and 5 it is clearthat different endometriosis patients do not react in the same way witheach of the 3 antigens. Therefore, if the pattern of reactivity ofendometriosis patient serum is considered for each of the 3 antigensthen this panel of markers makes the utility as a diagnostic test evenmore convincing. For example, 47 patients were evaluated with each ofthe ME-5, ME-2, and EPP2 antigens and the pattern of reactivity withantibodies in one or more specimens is summarized in Table 7. Takentogether, more that 83% of the sera tested contain antibodies whichreact with at least one of the ME-5, ME-2, or EPP2 antigens.Consequently if the three antigens were to be considered together as apanel for diagnostic testing then the frequency of antibodies inendometriosis patients that react with them is considerable.

TABLE 1 Overall pattern of ME-5 reactivity with 47 endometriosispatients. Serum ME-5 Rxn. DS01 + DS02 − DS03 + DS04 − DS05 + DS06 + DS07− DS08 − DS09 + DS10 + DS11 + DS12 + DS13 − DS14 + DS15 − DS16 + DS17 +DS18 − DS19 − DS20 − DS21 − DS25 − DS26 − DS27 + DS28 + DS29 + DS30 +DS31 + DS32 + DS33 + DS34 + DS35 − DS36 + DS37 − DS38 + DS39 + DS40 +DS41 − DS42 + DS43 + DS44 + DS45 + DS46 − DS47 − DS48 − DS49 − DS50 −Endometriosis patient serum specimens were obtained from a commercialsupplier (Diagnostic Support; Boston, MA) and evaluated for reactivitywith recombinant ME-5 antigen by line blot.

TABLE 2 Overall pattern of ME-5 reactivity with 24 control patient sera.Serum ME-5 Rxn. A1 − A2 − A3 ± A4 − A5 − A6 − A7 − A8 − A9 ± A10 − A11 −A12 ± A13 ± A14 ± A15 − A16 − A17 − A18 − A19 − A20 ± A21 − A22 ± A23 ±A24 − Control patient serum specimens were obtained from an internalblood draw and evaluated for reactivity with recombinant ME-5 antigen byline blot.

TABLE 3 Overall pattern of ME-2 reactivity with 47 endometriosispatients. Serum ME-2 Rxn. DS01 − DS02 − DS03 − DS04 − DS05 − DS06 + DS07− DS08 − DS09 − DS10 − DS11 − DS12 + DS13 − DS14 − DS15 − DS16 − DS17 +DS18 − DS19 + DS20 + DS21 + DS25 + DS26 + DS27 + DS28 + DS29 − DS30 +DS31 + DS32 − DS33 + DS34 − DS35 + DS36 + DS37 − DS38 + DS39 + DS40 +DS41 − DS42 + DS43 + DS44 + DS45 + DS46 + DS47 − DS48 − DS49 + DS50 +Endometriosis patient serum specimens were obtained from a commercialsupplier (Diagnostic Support; Boston, MA) and evaluated for reactivitywith recombinant ME-2 antigen by line blot.

TABLE 4 Overall pattern of ME-2 reactivity with 24 control patient sera.Serum ME-2 Rxn. A1 − A2 − A3 − A4 ± A5 − A6 − A7 − A8 − A9 ± A10 − A11 −A12 − A13 − A14 ± A15 − A16 − A17 − A18 ± A19 − A20 ± A21 − A22 − A23 −A24 − Control patient serum specimens were obtained from an internalblood draw and evaluated for reactivity with recombinant ME-2 antigen byline blot.

TABLE 5 Overall pattern of EPP2 reactivity with 90 endometriosispatients. Serum EPP2 Rxn. DS01 − DS02 − DS03 + DS04 − DS05 + DS06 + DS07− DS08 − DS09 − DS10 − DS11 − DS12 + DS13 − DS14 − DS15 + DS16 − DS17 −DS18 − DS19 + DS20 + DS21 + DS22 + DS23 − DS24 + DS25 − DS26 − DS27 +DS28 + DS29 + DS30 + DS31 + DS32 + DS33 + DS34 − DS35 + DS36 − DS37 −DS38 − DS39 − DS40 + DS41 − DS42 + DS43 + DS44 + DS45 + DS46 + DS47 +DS48 + DS49 − DS50 − BBI01 − BBI02 + BBI03 + BBI04 + BBI05 − BBI06 +BBI07 + BBI08 + BBI09 + BBI10 + BBI11 + BBI12 + BBI13 + BBI14 − BBI15 +BBI16 + BBI17 − BBI18 − BBI19 − BBI20 + BBI21 − BBI22 + BBI23 + BBI24 −BBI25 + BBI26 + BBI27 + BBI28 + BBI29 − BBI30 + BBI31 + BBI32 + BBI33 −BBI34 + BBI35 + BBI36 − BBI37 + BBI38 + BBI39 + BBI40 + Endometriosispatient serum specimens were obtained from a commercial supplier(Diagnostic Support; Boston, MA) and evaluated for reactivity withrecombinant EPP2 antigen by line blot.

TABLE 6 Overall pattern of EPP2 reactivity with 24 control patient sera.Serum EPP2 Rxn. A1 − A2 − A3 − A4 − A5 − A6 − A7 − A8 − A9 ± A10 − A11 −A12 − A13 − A14 − A15 − A16 − A17 − A18 − A19 − A20 ± A21 − A22 − A23 −A24 ± Control patient serum specimens were obtained from an internalblood draw and evaluated for reactivity with recombinant EPP2 antigen byline blot.

TABLE 7 Summary of the combined pattern of the panel of ME-5, ME-2, orEPP2 reactivity with 47 endometriosis patients. Serum Panel ReactivityDS01 + DS02 − DS03 + DS04 − DS05 + DS06 + DS07 − DS08 − DS09 + DS10 +DS11 + DS12 + DS13 − DS14 + DS15 + DS16 + DS17 + DS18 − DS19 + DS20 +DS21 + DS25 + DS26 + DS27 + DS28 + DS29 + DS30 + DS31 + DS32 + DS33 +DS34 + DS35 + DS36 + DS37 − DS38 + DS39 + DS40 + DS41 − DS42 + DS43 +DS44 + DS45 + DS46 − DS47 + DS48 + DS49 + DS50 + A total of 47endometriosis patient serum specimens were evaluated for reactivity witheach of the three antigens as shown in tables 1, 3, and 5. The overallreactivity of each of these patients with one or more of the markers issummarized.

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 44. A method for diagnosing endometriosis in a humansubject comprising the steps of: (a) detecting a test amount of anantibody that specifically binds to ME-5 (SEQ ID NO:3) polypeptide or apeptide comprising an epitope of ME-5, in a sample from the subject; and(b) comparing the test amount with a normal range of the antibody in acontrol sample from a subject who does not suffer from endometriosis,whereby a test amount above the normal range provides a positiveindication in the diagnosis of endometriosis.
 45. The method of claim44, wherein the sample comprises blood serum.
 46. The method of claim 44or 45, wherein the step of detecting comprises capturing the antibodyfrom the sample with an immobilized peptide where said peptide is ME-5or a peptide comprising an epitope of ME-5 and detecting capturedantibody.
 47. The method of claim 44 wherein said ME-5 polypeptide hasamino acid sequences substantially identical to SEQ ID NO:3 or anallelic variant thereof.
 48. The method of claim 44 wherein said ME-5polypeptide is produced by recombinant means.